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A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae

Author

Listed:
  • Yueping Zhang

    (Beijing University of Chemical Technology)

  • Juan Wang

    (Beijing University of Chemical Technology)

  • Zibai Wang

    (Beijing University of Chemical Technology)

  • Yiming Zhang

    (Beijing University of Chemical Technology)

  • Shuobo Shi

    (Beijing University of Chemical Technology)

  • Jens Nielsen

    (Beijing University of Chemical Technology
    Chalmers University of Technology
    Technical University of Denmark)

  • Zihe Liu

    (Beijing University of Chemical Technology)

Abstract

With rapid progress in DNA synthesis and sequencing, strain engineering starts to be the rate-limiting step in synthetic biology. Here, we report a gRNA-tRNA array for CRISPR-Cas9 (GTR-CRISPR) for multiplexed engineering of Saccharomyces cerevisiae. Using reported gRNAs shown to be effective, this system enables simultaneous disruption of 8 genes with 87% efficiency. We further report an accelerated Lightning GTR-CRISPR that avoids the cloning step in Escherichia coli by directly transforming the Golden Gate reaction mix to yeast. This approach enables disruption of 6 genes in 3 days with 60% efficiency using reported gRNAs and 23% using un-optimized gRNAs. Moreover, we applied the Lightning GTR-CRISPR to simplify yeast lipid networks, resulting in a 30-fold increase in free fatty acid production in 10 days using just two-round deletions of eight previously identified genes. The GTR-CRISPR should be an invaluable addition to the toolbox of synthetic biology and automation.

Suggested Citation

  • Yueping Zhang & Juan Wang & Zibai Wang & Yiming Zhang & Shuobo Shi & Jens Nielsen & Zihe Liu, 2019. "A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae," Nature Communications, Nature, vol. 10(1), pages 1-10, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-09005-3
    DOI: 10.1038/s41467-019-09005-3
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    Cited by:

    1. Wentao Zheng & Yuxuan Wang & Jie Cui & Guangyao Guo & Yufeng Li & Jin Hou & Qiang Tu & Yulong Yin & Francis Stewart & Youming Zhang & Xiaoying Bian & Xue Wang, 2024. "ReaL-MGE is a tool for enhanced multiplex genome engineering and application to malonyl-CoA anabolism," Nature Communications, Nature, vol. 15(1), pages 1-19, December.
    2. Liu, Zihe & Moradi, Hamideh & Shi, Shuobo & Darvishi, Farshad, 2021. "Yeasts as microbial cell factories for sustainable production of biofuels," Renewable and Sustainable Energy Reviews, Elsevier, vol. 143(C).
    3. Hui Liu & Pei Zhou & Mengya Qi & Liang Guo & Cong Gao & Guipeng Hu & Wei Song & Jing Wu & Xiulai Chen & Jian Chen & Wei Chen & Liming Liu, 2022. "Enhancing biofuels production by engineering the actin cytoskeleton in Saccharomyces cerevisiae," Nature Communications, Nature, vol. 13(1), pages 1-14, December.
    4. Dixit, Yatika & Yadav, Preeti & Sharma, Arun Kumar & Pandey, Poornima & Kuila, Arindam, 2023. "Multiplex genome editing to construct cellulase engineered Saccharomyces cerevisiae for ethanol production from cellulosic biomass," Renewable and Sustainable Energy Reviews, Elsevier, vol. 187(C).
    5. William M. Shaw & Lucie Studená & Kyler Roy & Piotr Hapeta & Nicholas S. McCarty & Alicia E. Graham & Tom Ellis & Rodrigo Ledesma-Amaro, 2022. "Inducible expression of large gRNA arrays for multiplexed CRISPRai applications," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    6. Qun Yue & Jie Meng & Yue Qiu & Miaomiao Yin & Liwen Zhang & Weiping Zhou & Zhiqiang An & Zihe Liu & Qipeng Yuan & Wentao Sun & Chun Li & Huimin Zhao & István Molnár & Yuquan Xu & Shuobo Shi, 2023. "A polycistronic system for multiplexed and precalibrated expression of multigene pathways in fungi," Nature Communications, Nature, vol. 14(1), pages 1-10, December.

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